Compelling work in the last decade has shown that transmembrane domains (TMDs), once thought to function solely as hydrophobic anchors, are central players driving cellular protein oligomerization, cell signaling, and regulation of channel function. However, the role of TMDs in viral glycoprotein stability and function remains poorly understood. During the previous funding period, our studies of the Hendra virus (HeV) fusion (F) protein identified key roles for TMDs in regulation of F protein stability, cellular trafficking, ad fusion function. Our novel studies utilizing sedimentation equilibrium (SE) analysis established that F protein TMDs efficiently trimerize separate from the rest of the protein. Altered TM-TM association was found for multiple functionally important mutations, implicating TM-TM interactions in regulation of F protein folding and function. The long-term goal of our research is to understand the molecular details and regulation of membrane fusion promoted by the paramyxovirus F proteins. The overall hypothesis of this proposal is that interactions of the TMD with itself and the lipid environment are critical for fusion protein stability and function, makin these interactions potential targets for antiviral therapeutics. To test this important hypothesis, we will pursue three specific aims. First, we will define key elements driving F protein TM-TM interactions including defining the role of a L/I heptad in TM-TM association and Hendra F protein stability. Second, we will determine how the TMD affects fusion triggering and promotion of membrane fusion, with a focus on the role of C-terminal -branched residues and a determination of the conformational changes that can occur when TM-TM interactions cannot be released. Third, we will dissect the role of the lipid environment in prefusion F stability in the context of cellular and viral membranes. These important experiments will provide critical new insight into the role of TMDs in regulation of F protein stability and fusion promotion.